Ribozymes are catalytic RNA molecules with endoribonuclease activity. The molecule can be divided into two different domains: the antisense portion, responsible for the recognition of and binding to the target RNA, and the catalytic domain which causes cleavage of the RNA. Modification of the antisense domain allows the redirection of the specificity of the molecule without affecting its catalytic properties. This makes it possible to down-regulate the amount of any given messenger RNA inside a cell, as long as the target sequence is known and contains potential ribozyme sites.
Ribozymes can be useful in solving a variety of problems, including viral disease infection and the process of malignant transformation. Although ribozymes are technically similar to antisense oligodeoxynucleotides (ODN) and antisense RNAs (AR), they have the advantage of catalytic activity that potentially allows a single molecule to destroy more than one target RNA. Despite this catalytic activity, a large molar excess of ribozyme over substrate is usually needed to get a significant reduction in the level of messenger RNA. Although this difficulty is possibly related to the intracellular localization of target RNA and ribozymes, another problem results from secondary structures formed by the target RNA and ribozyme which interfere with the necessary hybridization between the two structures.
In vitro cleavage studies utilize substrate and ribozyme transcripts which are usually shorter than their cellular counterparts. Specifically, expression vectors responsible for the synthesis of the ribozyme inside the cell contribute sequences upstream and downstream from the ribozyme motif to the final molecule, sequences which are not present in ribozymes synthesized in vitro. Nonproductive folding and non-specific hybridization of the ribozyme due to these extra sequences likely accounts for a portion of the loss of activity observed in vivo.
One possible solution to this problem is the use of chemically synthesized ribozymes. These molecules have the advantage of lacking the extra sequences, and they can be modified to make them more resistant to RNAses. However, when prolonged activity is needed, such chemically synthesized molecules require repeated administrations. Another possibility is to improve the activity of the ribozymes synthesized inside the cells by multiplying the number of catalytic molecules per cell or by increasing the activity of the individual molecules. Prior work has connected several ribozyme molecules within an expression vector (Chen et al., Nucleic Acids Research 20:4581-4589 (1992); Ohkawa et al,. PNAS USA 90:11302-11306 (1993); Weizsacker et al., Biochem. Biophys. Res. Commun. 189:743-748 (1992)) . One problem with these systems is that, after a certain number, the addition of more units to the construct does not correlate with an increase in the activity of the molecule, probably as a result of non-productive folding of the molecules which does not allow all of the units to interact with the target.
Others have used cis-acting ribozymes to reduce to a minimum the extra sequences upstream and/or downstream of the trans-acting motif (Chowrira et al., J. Biol. Chem. 269:25856-25864 (1994); He et al., FEBS Lett. 32:21-24 (1993); Taira et al., Nucleic Acids Research 19:5215-5130 (1991); Yuyama et al., Biochem. Biophys. Res. Commun. 186:1271-1279 (1992); Yuyama et al., Nucleic Acids Research 23:5060-5067 (1994)). Shotgun-type ribozyme expression vectors have also been designed in which tandems of transacting ribozymes are released by other (different) cis-acting ribozymes within the vector (Ohkawa et al., (1993)). However, the number of ribozyme monomers which can be incorporated in a single transcript is limited in this approach.